Collective invasion of squamous cell carcinoma requires LIMK activity in leading stromal fibroblasts. (A) The ability of squamous carcinoma cells (SCC) that retain epithelial markers to invade 3D matrix is dependent on stromal fibroblasts. LIMKi significantly inhibited SCC invasion in a dose-dependent manner. Photomicrographs indicate effects of LIMKi or Y27632 on SCC invasion. Bar, 100 µm. (B) Paths were allowed to be generated by fibroblasts, which were then removed by puromycin extraction before the addition of the SCC layer. Increasing LIMKi doses were either included only after path generation by fibroblasts was allowed to occur (SCCs targeted) or during fibroblast-mediated path generation (Fibroblasts targeted), as indicated. Significant inhibition of SCC invasion occurred specifically when fibroblasts were targeted, indicating that path generation but not path following required LIMK activity (averages ± SEM, n = 5). (C) To determine whether LIMK activity was required in fibroblasts for SCC invasion, two independent siRNA duplex pairs targeting LIMK1/2 were used to knock down expression. Relative to mock or NT siRNA-transfected cells, LIMK1/2 knockdown significantly reduced SCC invasion (averages ± SEM, n = 4). (D) To corroborate the dispensability of LIMK in SCC for invasion, two independent siRNA duplex pairs targeting LIMK1/2 were used to knockdown expression. Relative to mock or NT siRNA-transfected cells, LIMK1/2 knockdown did not significantly affect invasion, although Cdc42 knockdown did reduce invasion (averages ± SEM, n = 4). (E) Knockdown of MT1-MMP with three independent siRNA duplexes significantly reduced SCC invasion (averages ± SEM, n = 15).